In mammals, the pH of the extracellular compartment, including interstitial fluids and blood, is strictly regulated and maintained at a constant value of 7.4. Acid sensing is a specific kind of chemoreception that plays a critical role in the detection of nociceptive pH imbalances occurring, for example, in conditions of cramps, trauma, inflammation or hypoxia (Lindahl, Adv Neurol 1974; 4: 45)). In mammals, a population of small-diameter primary sensory neurons in the dorsal root ganglia and trigeminal ganglia (Bevan and Yeats, J Physiol (Lond) 1991; 433: 145) as well as central neurons (Varming, Neuropharmacol 1999; 38: 1875) express specialized pH-sensitive surface receptors activated by an increase of extracellular proton concentrations. Acid sensitivity of sensory as well as central neurons is mediated by a family of proton-gated cation channels structurally related to C. elegans degenerins (DEG) and mammalian epithelial sodium channels (ENaC). This invention relates to these Acid Sensing Ion Channels (ASIC) and specifically reports the discovery of novel class of receptors generated by the heteromultimeric assembly of two distinct ASIC subunits, namely ASIC2A (or BnaC1, or BNC1, or MDEG, or MDEG1) and ASIC3 (or hASIC3, or DRASIC) and uses thereof.
Tissue acidosis is associated with a number of painful, physiological (e.g. cramps) and pathological conditions (e.g. inflammation, intermittent claudication, myocardial infarction). Experimentally, similar painful events can be reproduced by infusing low pH solutions into skin or muscle. Furthermore, the prolonged intradermal infusion of low pH solutions can mimic the characteristic hyperalgesia of chronic pain. To further characterize the effects of protons and their relation to pain, low pH solutions were applied to patch-clamped central and peripheral sensory neurons. Inward currents were induced when pH was dropped to acidic values, providing evidence for the existence of proton-activated ion channels. Several types of native currents were observed in sensory neurons from rat and human trigeminal and dorsal root ganglia as well as central neurons: rapidly inactivating currents; non-inactivating currents; and biphasic currents displaying a rapidly inactivating current followed by non-inactivating sustained current. Other differences regarding ion selectivities were also reported. These results suggested the existence of a multigene family of proton-gated ion channels, implicated in neurotransmission and/or neuromodulation.
Cloned Proton-gated Ion Channels
The mammalian proton-gated cation channels have recently been cloned and named <<ASIC>> for Acid Sensing Ion Channels. Sequence analysis identifies them as members of the DEG/ENaC superfamily of ion channels. The putative membrane topology of ASIC receptors predicts two transmembrane spanning domains with both N- and C-termini in the intracellular compartment, as shown for the epithelial sodium channels. Four sub-classes of ASIC receptors have been identified:    1. ASIC1 ion channels display rapidly inactivating inward currents (Waldmann et al., Nature 1997; 386:173)    2. ASIC2 ion channels display slowly inactivating inward currents (Brassilana et al., J Biol Chem 1997; 272: 28819).    3. ASIC3 ion channels display biphasic inward currents with an initial rapidly inactivating component, followed by a sustained non-inactivating current (Waldmann et al., J Biol Chem 1997; 272: 20975; Babinski et al., J Neurochem 1999; 72: 51)    4. ASIC4 ion channels displaying similar properties as those of ASIC3 (Wood et al., WO9963081)
Other recently discovered ion channel subunits, BLINaC and INaC, appear to belong to the ASIC family but are not activated by protons and have not yet been shown to interact with other ASIC subunits (Sakai et al., J Physiol 1999; 519: 323, Schaefer et al., FEBS Lett 2000; In Press).
Families of ASIC Receptors Created by Alternative Splicing of mRNAs
A common feature of these ion channels is the existence of alternative splice variants, which display important functional differences. Indeed, the replacement of the first 185 amino acids of ASIC1 (hereinafter named ASIC1A) by a distinct new sequence of 172 amino acids generates a new channel, ASIC1B, which has similar current kinetics as ASIC1A but needs lower pH values for activation (pH50 of 6.2 and 4.5, respectively, for ASIC1A and ASIC1B). Also, it appears that ASIC1B is specifically expressed in rat dorsal root ganglia. A similar situation is also observed with rat ASIC2 (hereinafter named ASIC2A), where the replacement of the first 185 amino acids by a distinct new sequence of 236 amino acids generates another ASIC ion channel subunit, ASIC2B. When expressed alone as a homomultimer in mammalian cells or Xenopus oocytes, ASIC2B does not appear to be activated by low pH solutions. ASIC3, which has been identified in human, also appears to exist in various forms. Indeed, DRASIC is an ASIC3-like channel identified in rat, which displays 85% identity with the human ASIC3 sequence and has similar biphasic current kinetics. However, important differences regarding tissue distribution, ion selectivities and pH50 suggest that DRASIC might not be the human orthologue of ASIC3 but rather a different subtype. Furthermore, the existence of two 3′ splice variants of human ASIC3 (ASIC3B and 3C, sequences submitted to GenBank) have been reported but differences in function have yet to be documented. Alternative splicing, therefore, appears like an important mechanism for increasing the diversity of ASIC receptors, which most probably assume critical roles in the nervous system, such as neurotransmission, nociception or mechanosensation (see below).
Families of ASIC Receptors Created by Heteromultemeric Associations
In general, functional ion channels are complex structures comprised of several individual components, referred) to as subunits. The number of subunits depends on the type of ion channel and subunits can either be all identical (homomultimeric channels) or include a combination of several different subtypes (heteromultimeric channels). For example, Epithelial sodium Channels (ENaC), which belong to the same gene family as ASIC receptors, are comprised of at least three different subunits, namely αEnaC, βEnaC and γEnaC (Canessa et al., Nature 1994; 367: 463). Although cloned ASIC receptors have mostly been characterized in vitro in their homomultimeric form, the analogy with EnaCs raises the possibilty that ASIC subunits might also associate in various combinations to generate novel channels with distinctive properties. Indeed, heteromultimeric ASIC channels might account for some of the native proton-gated currents still not explained by any of the homomultimeric ASICs cloned to date. Examples of such native currents are the sustained non-desensitizing currents seen at pH 6 (Bevan and Yeats, J Physiol 1991; 433: 145). Furthermore, the discovery of the proton-insensitive ASIC2B (or MDEG2) suggests that it may function as an accessory subunit. Indeed, the first evidence for heteromultimeric ASIC receptors came from coexpression studies featuring rat ASIC2B either with ASIC2A or with ASIC3. Channels created by ASIC2A and ASIC2B appear to be slightly more sensitive to pH, while inward currents carried by ASIC2B+ASIC3 channels are apparently less sodium selective than the homomultimeric ASIC3 currents (Lingueglia et al., J Biol Chem 1997: 272: 29778). However, no biochemical evidence of interaction has been reported to date for any ASIC subunits. Furthermore, other coexpression experiments with different subunits suggest that not all subunit combinations yield novel functional channels. Thus, the composition and functional characteristics of heteromultimeric ASIC channels are therefore unpredictable.